So, I've treated both of my primers separately with T4 PNK using folowing protocol:
1 µl of 100 µM primer + 1 µl of T4 ligase buffer + 1 µl of PNK (10000 U/ml) + 7 µl NF water in each of eight PCR tubes ==> incubate at 37°C for 1 hour ==> heat inactivation (next day) by 20 min. incubation at 80 °C.
Then, I tried using them to PCR amplify a 5.5 kb amplicon with Q5 polymerase, and also tried the exact same with non-phosphorylated primers as a control. For the PNK-treated primers, the PCR fails, but for the non-phosphorylated primers it works just fine. Does anyone know what can be the cause or how to solve this?
It is possible that your phosphorylation reagent contains a nuclease or that your process inactivates ssDNA. You could try running the phosphorylation reaction exactly as before ( including deactivation) but without the PNK and then compare the unreacted primer against the dummy reacted primer to test the reagents and procedure. I assume that you have adjusted the dilution of post PNK primers to ensure that the primer concentration of primer both prosphorylated and not phosphorylated are both the same in the pcr reaction given that there has been some dilution during the PNK reaction
I would recommend using T4 PNK for your PCR product, not for your primers. I always do like that (>20 years). By the way, many T4 PNK buffers are compatible with T4 ligases, thus no additional purification steps are needed after PNK.
Hi Paul Rutland , thanks for your answer ! That seems like a good idea, so I'll be trying it tomorrow. Concerning the primer concentration, I'm using 1 µl of 100 µM primer stock in each PNK reaction of 10 µl, thus getting 10 µM primer solution. I have also diluted my non-treated primers from 100 to 10 µM as in my lab we always work with a starting concentration of 10 µM for our primers. So, since I use the same volume of primers (1.25 µl per primer in a 25 µl PCR reaction), the concentration in the PCR reaction should also be the same for both treated and untreated primers.
Hi Ruslan Kalendar , for both treated and untreated primers, I use a concentration of 10 µM and add 1.25 µl of both Fw and Rv primer to a PCR reaction of 25 µl. I have used the same calculations for the PCR mixture for both treated and untreated primers, so if I made a calculation error I would also not expect amplification using the untreated primers right?
Hi Pavel Uvarov , thanks for your answer! I might try it that way though I thought I had read somewhere that it PNK doesn't work very efficiently on dsDNA molecules. I'm not sure what you mean with your second sentence; do you mean I that using T4 PNK buffer might be more effective than T4 ligase buffer?
Hi Ben Neven, I may also have seen somewhere that the efficiency of T4 PNK is lower on dsDNA compared to ssDNA, but cannot find this info now. Yet, this efficiency is still very high and PNK works very well on dsDNA. I have never experienced problems with subsequent ligation of PCR products. Clarification to my previous answer - many companies provide T4 ligase buffer compatible with T4 PNK. So, PNK and ligation reactions can be done in the same T4 ligation buffer (PNK first and then continue with ligation).
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